The actin cytoskeleton participates in diverse cellular functions including cellular organization, polarized cell growth, membrane traffic, cell shape changes, cytokinesis and cell motility. These functions require precise control of the assembly, dynamics and organization of the actin filaments that constitute the core of the actin cytoskeleton. Defects in proper actin regulation can contribute to human disease including cancer cell metastasis, Wiskott-Aldrich Syndrome, and sickle cell crisis to name a few. Our lab seeks to understand how actin associated proteins of S. cerevisiae regulate the assembly and dynamics of actin filaments and how this regulation contributes to cell function. The focus is on three novel regulators of actin dynamics: Actin Interacting Protein 3 (Alp3), Actin Interacting Protein 1 (Alp1), and Old Yellow Enzyme (Oye2). Aim#1: An in vitro biochemical analysis of Aip3 activity will be undertaken to understand how Aip3p cooperates with other cellular proteins to coordinate cell-cycle controlled, polarized actin assembly. This will be complemented by genetic analyses designed to uncover the networks of genes that cooperate with AlP3 to regulate polarization of the actin cytoskeleton. Aim #2: Aip1 is a novel actin filament severing protein whose activity is constrained to filaments decorated with cofilin, another actin binding protein. Through mutagenesis we have begun to uncover the structure/function relationships between domains of Aip1 and cofilin. This has led to hypotheses concerning how Aiplp negatively auto-regulates its severing activity and possible mechanisms for the severing reaction. We will test these hypotheses by identifying protein interaction defects of mutant alleles and correlating those defects with biochemical defects in Alp1 regulation and filament severing. Genetic analysis will be used to uncover the genes that interact with an aip1delta allele, these will be tested against our battery of aip1mutant alleles to define a genome level, genetic and biochemical structure/function map of ALP1.Aim#3: Preliminary analysis of oyedelta strains suggests that Oye2p regulates reduction of an intramolecular disulfide bond in actin. This form of actin oxidation in red blood cells contributes to sickle cell crisis by reducing the dynamic behavior of actin filaments. We will test this comparison by quantifying the amount of oxidized actin that accumulates in oyedelta yeast strains and by directly measuring reduction of red blood cell actin by Oye2p. Genetic analysis will be used to define those genes and pathways that require proper redox regulation of the actin cytoskeleton. These experiments will help to establish the yeast oyedelta strain as a model system for human disease states (such as sickle cell crisis) that are affected by actin oxidation.